The significance of endocrine-disrupting compounds (EDCs) in the environment has only recently come to the forefront of scientific research, policy debates, water utilities management and public awareness. EDCs have the ability to interfere with the normal functioning of the endocrine system of humans and other animals. Numerous chemicals are included in the class of compounds known as EDCs, and exposure is widespread. These compounds are found in a variety of environmental matrices (e.g., marine and freshwater systems, sediment, soil), transported there primarily through sewage effluent discharge and recycling of sewage sludge for topical fertilizer use. This transport to the environment serves as the primary route of exposure for aquatic and terrestrial organisms living there. Furthermore, these compounds are also found in consumer products, food and drinking water--which serve as the exposure source for human beings. Multiple examples of endocrine disruption have been documented in humans and animals, and certain EDCs have been implicated in each case. The future of public and environmental health will depend upon mitigating the effects of these chemicals. This purpose of this dissertation is to provide an initial understanding of EDC occurrence in the Tampa Bay region of south Florida, and to complement the existing body of EDC research with regards to marine systems. It focuses on estrogenic EDCs, specific compounds which target the estrogen axis of the endocrine system. Six estrogenic EDCs were chosen based on their documented prevalence in the environment, prevalence in sewage, and for their suspected endocrine-disrupting effects: estrone, 17β-estradiol, estriol, 17α-ethinylestradiol, bisphenol-A and nonylphenol. These compounds were verified to be amenable to and detectable by gas chromatography-mass spectrometry analysis. Since the occurrence of EDCs in aquatic environments of the Tampa Bay region had not been previously characterized, the initial phase of the research focused on quantification of the six estrogenic EDCs in Tampa Bay area water, sediment, and sewage influent and effluent. All targeted EDCs were present in 89% of sewage samples, while 100% of the samples contained at least one or more EDCs. The concentrations of EDCs in marine aqueous and sediment samples tended to decrease with increasing distance from the wastewater treatment plant discharge site. The ubiquitous presence of these estrogenic EDCs in the Tampa Bay area is cause for concern with respect to endocrine disruption in local terrestrial and aquatic wildlife. Since the Tampa Bay region is home to a wide variety of marine organisms, constant exposure to EDCs could result in ecosystem-level effects, as these compounds can impair reproductive fitness and lead to other adverse health effects. This research also served to enlarge the existing scientific literature on EDC occurrence, as many marine and freshwater systems continue to be characterized globally. The very basis for expecting to find EDCs in the Tampa Bay area had come from the fact that the main source of environmental contamination is typically the effluent discharge from area wastewater treatment plants. Conventional wastewater treatment plant processes are designed to reduce the amount of organic matter, pathogens and nutrients from the incoming influent. However, the processes are not as effective in removing micropollutants, including EDCs. These compounds notoriously evade traditional wastewater treatment technologies and are found even in tertiary-treated effluent. For this reason, the second phase of the research assessed an electro-chemical technique for the removal of the same six EDCs. The removal technique was tested on a laboratory scale and has a commercial-sized counterpart which can be integrated at the level of the wastewater treatment plant. In order to test the removal efficiency, samples of influent and tertiary-treated effluent were spiked with the six EDCs. The mean concentration of each EDC component was statistically lower after treatment (removal range = 42% - 98.2%), demonstrating the effectiveness of this electro-chemical process for EDC removal from both raw and treated sewage. The significance of the results lies in the fact that if this method is implemented, then future wastewater treatment plant effluent discharge (similar to that of the Tampa Bay region) could be less impacted by EDCs and therefore cleaner for the environment into which it is being discharged. For the final phase of the research, the use of computational techniques to simulate human endogenous estrogen binding to its receptor was started as a foundation for future models to eventually predict endocrine-disrupting potential of different chemical compounds. We built an estradiol-human estrogen receptor model, and used molecular dynamic simulations to determine the binding free energy. The calculated total binding free energy of estradiol bound to the ligand binding domain of the human estrogen receptor was found to be -16.85 kcal/mol, which is in range of the experimental value of -12.40 kcal/mol. Humans are chronically exposed to low doses of EDCs every day, which makes endocrine disruption a considerable public health issue. Human exposure to EDCs is completely different from marine organism exposure, but the adverse effects are no less significant. The successful completion of this model serves as a platform for 1. Testing the human model against endocrine-disrupting compounds, 2. Subsequent models that will be developed for different species, including marine species important to Tampa Bay. Substantial data exist regarding the exposures and health risks associated with EDCs in humans and wildlife on a global scale. As the pressing issues of climate change and carbon emissions are at the top of the list of environmental concerns, it is important to note that mitigating the effects of EDCs should not be overlooked and will be an important responsibility of regulatory agencies in the near future.

Following the BP Deepwater Horizon oil spill event, there was a critical need to assess the effects of the oil and dispersant chemicals on the coastal pelagic fish complex in the Gulf of Mexico and the Florida Straits. The objective of this study was to determine if spilled crude oil and dispersant chemicals have posed an ecological risk to the coastal pelagic fish complex through the detection of vitellogenesis. Crude oil containing polycyclic aromatic hydrocarbons (PAHs), dispersant chemicals and other estrogen-mimicking compounds are suspected to induce vitellogenin production in male and immature female fish, normally only produced by sexually mature females. Blood plasma and surface mucus were collected from wild-caught adult and juvenile males and females from as many representative coastal pelagic species as possible (including yellowfin tuna Thunnus albacares and swordfish Xiphias gladius). To create a control for this experiment, crevalle jacks (Caranx hippos) and lookdowns (Selene vomer) were injected with estradiol-17β (10 μg/g body weight) into the peritoneal cavity to induce vitellogenesis, regardless of sex or reproductive stage. The mucus and blood plasma of each injected fish was collected 7 to 11 days post-injection. Mucus and blood plasma samples of wild-caught and experimentally-injected fishes were separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, stained with a phosphoprotein specific fluorescent dye (Pro-Q Diamond®), and visualized through ultraviolet transillumination. Vitellogenin was visibly detectable in the mucus collected from the control-injected fish, suggesting a disruption in the endocrine system as a result of estrogen exposure (estradiol-17β). However, there were no elevated levels of vitellogenin detected in any wild-caught fish mucus or blood plasma samples, indicating no vitellogenesis. From this, we infer that there has been no detectable endocrine disruption to the sampled coastal pelagic fish complex in the Gulf of Mexico two years after the start of the spill.

Fluoxetine (FLX), the active ingredient of Prozac™, is a member of the selective serotonin reuptake inhibitor (SSRI) class of anti-depressant drugs and is present in aquatic environments worldwide. Previous studies reported that FLX is an endocrine disruptor in fish, bioconcentrating in tissues including the brain. Evidence implicates that serotonin influences the activity of the hypothalamo-pituitary-interrenal (HPI) stress axis, thus exposure to FLX may disrupt the teleost stress response. This study examined in vitro cortisol production in rainbow trout (Oncorhynchus mykiss) head kidney/interrenal cells exposed to FLX and 14C-pregnenolone metabolism in head kidney microsome preparations of FLX-exposed trout. Results indicated that cells exposed in vitro to increasing concentrations of FLX had lower cortisol production and cell viability (versus control) and microsomes isolated from trout exposed to 54 μg/L FLX had higher pregnenolone metabolism versus those of control and low FLX-exposed (0.54 μg/L) trout.

Parmi les outils existants pour l'évaluation de la qualité des milieux aquatiques, les biomarqueurs permettent de détecter précocement l'exposition et/ou les effets d'une contamination. Toutefois, en raison du manque de connaissance fondamentale de leur régulation au niveau moléculaire, peu de biomarqueurs ont été spécifiquement développés chez les invertébrés, alors que ces derniers représentent 95% de la biodiversité animale. Grâce aux récentes avancées technologiques dans le domaine du séquençage de l'information génétique et protéique, la découverte de nouvelles protéines chez ces organismes est à présent possible. Centré sur l'utilisation d'une espèce d'intérêt en écotoxicologique, l'amphipode d'eau douce Gammarus fossarum, et sur la problématique de la perturbation endocrine, ce travail doctoral a pour objectif de découvrir de nouvelles protéines impliquées dans la reproduction du gammare et de proposer des biomarqueurs spécifiques de reprotoxicité. Par une première approche protéogénomique, un important catalogue de protéines a été généré. Puis, afin d'identifier de nouvelles protéines impliquées dans la reproduction, une comparaison entre les protéomes des tissus reproducteurs mâle et femelle a été réalisée, suivi de l'étude de la dynamique du protéome au cours de différents processus physiologiques : ovogénèse, embryogénèse et spermatogénèse. Enfin, une dernière expérience sur des organismes mâles exposés à différents perturbateurs endocriniens a permis d'identifier différents candidats biomarqueurs de reprotoxicité. Cette étude ouvre la voie à des développements rapides de biomarqueurs spécifiques d'une espèce animale d'intérêt en écotoxicologie / Among the tools for assessing biologic quality of aquatic ecosystems, biomarkers are ideally suited for the early detection of contaminant exposure and/or effects. Yet, because of the lack of fundamental knowledge of their molecular regulation, few specific developments have been carried out on invertebrates even though they account for more than 95% of animal biodiversity. Thanks to recent technological advances in nucleic and proteic information sequencing, discovery of new proteins is now possible for these organisms. Focussed on the use of an ecotoxicologically relevant species, the freshwater amphipod Gammarus fossarum and the problem of endocrine disruption, this doctoral thesis aims to discover new proteins involved in gammarid reproductive function and to propose specific biomarkers of endocrine disruption. With a first proteogenomic approach, an important protein catalogue was generated. Next, to identify proteins involved in gammarid reproduction, male and female reproductive tissue proteomes were compared, followed by the study of proteome dynamics in several physiological processes: oogenesis, embryogenesis and spermatogenesis. Finally, a last experiment on male gammarids challenged with different endocrine disrupter chemicals identified several candidate biomarkers of reprotoxicity. This study paves the way for quick developments of specific biomarkers for organisms of interest in ecotoxicology

Depuis plusieurs décennies, le rôle des expositions environnementales dans l’apparition des anomalies de la reproduction suscite l’intérêt de la communauté scientifique. La période prénatale est une phase critique du développement et l’exposition à des substances chimiques au cours de la grossesse pourrait avoir des conséquences sur la santé de l’enfant et de l’adulte. De nombreuses études chez l’animal ont mis en évidence que diverses molécules chimiques étaient toxiques pour la reproduction et le développement et que certaines d’entre elles étaient capables d’interférer avec le système endocrinien. Chez l’Homme, les effets de l’exposition prénatale à des substances susceptibles d’altérer l’équilibre hormonal du fœtus restent mal documentés. L’objectif de ce travail de thèse est d’étudier l’effet de l’exposition prénatale à certains polluants chimiques en s’intéressant à deux indicateurs d’anomalie du développement du système reproducteur à la naissance : les niveaux d’hormones sexuelles et les malformations congénitales de l’appareil génital du garçon. Ce travail s’appuie sur les données issues de cohortes mère-enfants françaises ayant recruté des femmes enceintes au cours de leur grossesse et pour lesquelles des prélèvements biologiques ont permis la mesure de l’exposition. Les classes chimiques plus spécifiquement étudiées dans cette thèse comprennent les polluants organiques persistants (polychlorobiphényles, pesticides organochlorés et retardateurs de flamme bromés) et les éthers de glycol, une classe de solvants oxygénés. Les études menées mettent en évidence des effets sur le développement du système reproducteur, potentiellement associés à des mécanismes de perturbation endocrinienne, en lien avec l’exposition prénatale aux deux familles de polluants étudiées. Des modifications des niveaux d’hormones sexuelles sont observées en association avec l’exposition prénatale à divers polluants organiques persistants. L’exposition prénatale à certains éthers de glycol est associée à une augmentation du risque d’hypospade ainsi qu’à des modifications des niveaux d’hormones sexuelles. Ces résultats mettent en évidence les effets d’expositions prénatales à des polluants chimiques ubiquitaires, sur le système endocrinien du fœtus. / From last decades, researchers had increased interest about the impact of environmental exposure on reproductive impairments. The fetal life is a crucial period of development and exposure to chemical during gestation may lead to adverse health outcomes at birth or later in life. Several toxicological studies have reported that some chemicals are reproductive and developmental toxicants and that some of them are able to interact with the endocrine system. In humans, evidence about the endocrine effects of these molecules is limited. The aim of this thesis is to study the effect of prenatal exposure to chemicals on two endocrine-sensitive endpoints at birth: sex hormone levels and congenital anomalies of the genitalia. This work is based on data collected in two French mother-child cohorts that included pregnant women during pregnancy and collected biological samples to perform exposure assessment. Two chemicals classes are studies including persistent organic pollutants (polychlorinated biphenyls, organochlorine pesticides, and polybrominated diphenyl ethers) and glycol ethers, a class of oxygenated solvent. The studies conducted show effects of prenatal exposure to both classes of chemicals on endocrine-sensitive endpoints related to reproductive health. Modifications of sex hormone levels are observed in association with exposure to various persistent organic pollutants. Prenatal exposure to some glycol ethers is associated with an increased risk of hypospadias and with modifications of sex hormone levels. These results highlight the effect of prenatal exposure to ubiquitous chemicals, on the endocrine system of the fetus.

No description available.

Estrogenic chemicals are common pollutants in wastewater and current methods used in wastewater treatment processes are not typically effective in removing these compounds. Thus, many estrogenic chemicals, as well as other pharmaceuticals, are detected in drinking water supplies, contributing to human exposures. This concerning public health situation has led some green chemists to investigate the feasibility and efficacy of tetra-amido macrocyclic ligands (TAML), which have been proposed to be environmentally friendly catalysts that can be used to treat wastewater. TAML works by catalyzing the oxidation of micropollutants, breaking pollutants down into substances that can be assimilated by bacteria. These breakdown products are anticipated to no longer pose an environmental or health concern. Prior to their use in environmental matrices, TAML must first be evaluated to ensure that these compounds pose a low environmental and human-health hazard. Here, we exposed adult female mice to one of three doses of New-TAML 7 (NT7), or an estrogen receptor agonist (ethinyl estradiol, EE2) or an androgen receptor antagonist (flutamide), for two weeks prior to mating, throughout pregnancy, and throughout the lactational period. Male mice used for breeding purposes, the F0 males, were also exposed to the drinking water during the 2-week mating period. Following this acute exposure, the F0 males were necropsied and evaluated for signs of overt toxicity and repro-endocrine disruption. There was a statistically significant decrease in the F0 males spleen weight. Male offspring, the F1 generation, were evaluated at weaning (postnatal day 21), in puberty (postnatal day 32), and in adulthood. We first evaluated organs known to be sensitive to toxic effects. In male mice developmentally exposed, we have discovered that all treatments decreased spleen size at postnatal day (PND) 21 but not in early adulthood. At PND 16, no significant effects were observed. At PND21, no effects were observed on the weight of the kidney, liver, testes, or seminal vesicles; however, spleen weight was significantly decreased in the positive controls and the mid-NT7 group. In early adulthood, no effects have been observed on organ weights of the flutamide group; however, the size of both testes is significantly affected by the mid-NT7 dose and the EE2 treatment. By six months of age, there were no statistically significant effects, although this is likely in part due to inadequate statistical power. We also evaluated the effect of NT7, EE2, and flutamide on the male mouse mammary gland. Because of the conserved role hormones play in mouse development, a mammary gland is a valuable tool for identifying endocrine disruptors. We found that EE2 increased the size and number of branching points starting at PND21 and continuing until nine weeks of age. TAML had little effect on the morphology of the male mammary gland, with the exception of increased ductal branching at PND21. This, along with the change in testes weight, may suggest TAML can affect estrogen-mediated outcomes; many of our observed effects warrant additional study.

No description available.

No description available.

Steroid hormones confer biological activity to effluents of wastewater treatment plants (WWTPs). The occurrence of estrogen and androgen hormones in addition to their biological effects in the environment have been widely studied and there is a growing consensus that mixtures of steroid hormones; albeit at low ng L-1concentrations, lead to endocrine disruption in some aquatic organisms. These mixtures may also be influenced by the contributions of synthetic estrogens and androgens, which may display either additive or antagonistic activity. In order to measure the ability of a single compound, or complex mixture to influence the function of estrogenic or androgenic signaling pathways bioassays are used. Most commonly, these tests are in vitro and may quantify the ability of a compound to bind and/or (in) activate the steroid receptors. Two commonly used bioassays for estrogenicity detection are the Yeast Estrogen Screen (YES) and the E-Screen assay. The Yeast Androgen Screen (YAS) is commonly used to measure androgenic activity. The yeast (Saccharomyces cerevisiae) are genetically transformed and express either the human Estrogen Receptor (ER) or Androgen Receptor (AR), and contain Estrogen or Androgen Responsive Elements (ERE/ARE) and Lac Z reporting plasmids. Once the receptors become activated, beta-Galactosidase is secreted into the assay medium and the level of beta-Galactosidase secretion relates to the estrogenicity or androgenicity of the sample tested. Due to its simplicity and the moderately fast assay time, the YES and YAS are commonly used assays in the analysis of complex mixtures to identify the major contributors to both estrogenic and (anti)-androgenic activity in environmental water. The effect directed approach combines both chemical methods and bioassays in a chemical fractionation scheme that is directed by the bioassays. In order to confirm the identity of the key contributors, it is important to compare the biological activities that are calculated from the concentrations of the identified hormones (given their individual biological responses) and the total biological activity measured through the use of bioassays, Equation 1. RPsCs+ RP2C2+ ...+RPnCn = IEQ (1) where Cn is the concentration of the nth mixture constituent, RP is the relative (estrogenic or androgenic) potential of the nth mixture constituent as determined in the bioassay, and IEQ is the estimated total induction equivalent concentration of the mixture by chemical methods. Cs and RPs represents the concentration and relative potential of a standard compound respectively. Agreement between the chemically and biologically derived IEQs means that the major contributors to the biological effect have been successfully identified. However, the biological assays measure the contribution of additive, antagonistic and synergistic activity in the mixture; therefore, the biologically derived IEQs represent the net biological activity. Chemical methods are unable to predict these interactions and as such the result of the concentration addition (CA) approach (Equation 1) is often inconclusive and suggestive of interacting components. An interaction model that can estimate the net biological activity of a mixture from the concentrations of individual mixture constituents (chemical methods) is thus necessary. An interaction model that combines both the relative potential (RP) as well as the interaction index (γ) in a parameter called aRP was developed. The aRP is defined by Equation 2 and is used similarly to the CA approach, Equation 3. aRP = interaction index-1RP (2) aRPsCs+ aRP2C2+ ...+aRPnCn = IEQ (3) The aRP can be calculated for any nth mixture constituent by measuring the degree to which the mixture components altered the activity of the standard and assessing those changes as a function of mixture ratios. The interaction method was validated using a mixture of testosterone, with two anti- androgens, di-n-butyl phthalate, and bisphenol A in the YAS. Mixtures of 17ß;-estradiol, estriol, 17α-dihydroequilin and di-n-butyl phthalate were evaluated in the YES assay. Using Equation 3 the net estrogenic and androgenic activity of the mixtures was estimated. There was a significant improvement over the CA based approach in Equation 1. Overall, in 24 out of 32 mixtures tested there was no significant difference between the aRP and observed responses. Large percent errors were observed in the CA model, particularly when the proportion of antagonists was high as the CA model tended to over-predict the responses. On the contrary, only two aRP model predictions exceeded 50% error. Risk assessors should use the CA model with caution as it could over-predict biological responses and an alternative approach such as the aRP model could be used. In this regard, a database of aRP values for identified antagonistic/synergistic compounds could be assembled and estimations of biological activity could be made using these aRP values. The aRP interaction model could also be used to provide fundamental understanding to the behavior of the constituents in a complex mixture. Although the interaction model presented may account for possible deviations from additivity in environmental mixtures, predictions of mixture effects may be complicated by matrix interferences. In this regard, a sensitive bioassay; such as the E-Screen, which is capable of detecting concentrations as low as 0.27 ng L-1 of 17β-estradiol equivalents is beneficial. However, one major drawback to the E-Screen assay is the 6-day analysis time. In order to maintain the sensitivity of the assay and reduce the analysis time, Fourier Transform Infra-red Imaging Spectroscopy (FT-IRIS) was used to probe the bio-molecular level events that occur in single cells prior to a detectable response in cellular proliferation. The investigation revealed that changes occur on the sub-cellular level at 48-hours after incubation which are comparable to the 6 day E-Screen responses (Pearson R = 0.978). The FT-IRIS response appears to be due to the increase in mucins which are known to play a role in cellular signaling and proliferation. The EC50 values for the E-screen and FT-IRIS assay were 2.29 and 2.56 ppt respectively, indicating that the molecular changes, which are observed at the single cell level using FT-IRIS, are reflective of physiological changes that are observed as the cell population responds to 17ß-estradiol. The study indicates that sophisticated imaging and microscopy techniques such as FT-IRIS may play a role in environmental bio-analytical methods. / Civil Engineering